The Adipose Tissue Core will provide a wide variety of cost-effective services for the study of[unreadable] human and mouse adipose tissue. Services will include techniques of cell separation and sorting,[unreadable] histology, molecular biology and protein chemistry. Specifically, freshly isolated human and mouse[unreadable] fat samples will be subjected to collagenase digestion (under conditions least likely to alter gene[unreadable] expression) to separate primary adipocytes from the stromal vascular fraction. Adipose tissue[unreadable] macrophages will subsequently be separated from the stromal vascular fraction with antibodycoated[unreadable] magnetic beads. Gene expression will be analyzed in whole adipose tissue and its cellular[unreadable] components using quantitative 'real-time' reverse-transcriptase polymerase chain reaction (rt-PCR,[unreadable] Roche LightCycler). Quantification of RNA copy number will be derived from plasmid standard[unreadable] curves for every gene of interest. Analysis of gene expression will be complemented by analysis of[unreadable] protein expression of key adipocyte secretory products by Western blotting. Additionally, Western[unreadable] blotting will be used to study the activation of various components of the insulin signaling pathway[unreadable] in adipose tissue sampled under a variety of in vivo conditions. Quantitative assessment of[unreadable] macrophage infiltration into adipose tissue will be performed by two alternative methods:[unreadable] quantitative rt-PCR (using specific macrophage markers) and Fluorescence Activated Cell Sorting[unreadable] (FACS) analysis. Since many of the above techniques require freshly obtained adipose tissue, the[unreadable] concentration of these techniques in one Core should favor efficiency and minimize handling.[unreadable] The following are several additional, highly specialized techniques offered by the Core. Adipocyte[unreadable] size and number will be quantified following osmium fixation of adipose tissue samples. Serial[unreadable] biopsies of rat adipose tissue will be performed in vivo by Dr. Vasselli. Lipoprotein lipase activity will[unreadable] be assayed in frozen adipose tissue samples by Dr. Johnson. Recombinant adipokine production[unreadable] will be provided by Dr. Lawrence Shapiro.